bims-proteo Biomed News
on Proteostasis
Issue of 2025–10–26
forty-six papers selected by
Eric Chevet, INSERM
  1. A paradoxical role of autophagy: promoting protein aggregates and hindering rejuvenation in a Caenorhabditis elegans L1 arrest model.
  2. Global analysis of translocon remodeling during protein synthesis at the ER.
  3. A structural basis for chaperone repression of stress signaling from the endoplasmic reticulum.
  4. The many faces of the GID/CTLH E3 ligase complex.
  5. Heat shock induces silent ribosomes and reorganizes mRNA turnover.
  6. The mitochondrial disulphide relay substrate FAM136A safeguards IMS proteostasis and cellular fitness.
  7. Optineurin is an adaptor protein for ubiquitinated substrates in Golgi membrane-associated degradation.
  8. Navigating the landscape of protein folding and proteostasis: from molecular chaperones to therapeutic innovations.
  9. ATG9 assists lipid replenishment for lysosome membrane repair.
  10. Delayed protein translocation protects mitochondria against toxic CAT-tailed proteins.
  11. In situ characterization of mitochondrial Hsp60-Hsp10 chaperone complex under folding stress.
  12. Small-molecule degron mimetics for targeted protein degradation.
  13. The Proteostasis Network is a Therapeutic Target in Acute Myeloid Leukemia.
  14. Computational design and evaluation of optimal bait sets for scalable proximity proteomics.
  15. SDF2 and SDF2L1 are essential co-factors of DNAJB11 for Polycystin-1 processing.
  16. ITCH regulates Golgi integrity and proteotoxicity in neurodegeneration.
  17. RNase MRP subunit composition and role in 40S ribosome biogenesis.
  18. Reticulophagy receptor FAM134C restrains BMP receptor signaling.
  19. Mammalian and Bacterial Adaptors Function as Co-Disinhibitory Pairs to Activate the E3 ubiquitin ligase WWP2.
  20. The Drosophila ZER1 homolog interacts with ref(2)P to regulate autophagy and Keap1-cnc/NFE2L2/Nrf2-mediated oxidative stress.
  21. Plasma membrane-endoplasmic reticulum coupling probed with genetically-encoded voltage sensors.
  22. Functional landscape of ubiquitin linkages couples K29-linked ubiquitylation to epigenome integrity.
  23. On-genetic inactivation of caspase-3 and P53 increases cancer cell fitness by PDIA4 redistribution.
  24. Enhanced hybridization-proximity labeling discovers protein interactomes of single RNA molecules.
  25. TXNIP mediates LAT1/SLC7A5 endocytosis to limit amino acid uptake in cells entering quiescence.
  26. Targeted protein degradation of HSP90 and associated proteins for cancer therapy via PROTACs and beyond.
  27. Physics-based evolution of transmembrane helices reveals mechanisms of cholesterol attraction.
  28. Nir2 crystal structures reveal a phosphatidic acid-sensing mechanism at ER-PM contact sites.
  29. ER stress induced mitochondrial dysfunction drives Treg instability in coronary artery disease.
  30. Unveiling Sec31: The regulator of COPII vesicle-dependent secretion and beyond.
  31. J-domain proteins cooperate with Hsp70 to drive multiphase separation of RNA-binding-deficient TDP-43.
  32. Tudor-based proteomic strategy pan-specifically enriches and identifies protein arginine methylation.
  33. Human clearance systems have a layered architecture across tissues and cell types that supports varied proteome compositions.
  34. Establishment of an imaging-based screening pipeline for the identification of human ribosome biogenesis inhibitors.
  35. CIRCpedia v3: an interactive database for circular RNA characterization and functional exploration.
  36. Adipose cullin 3 mediates the antiobesity effect of pan neddylation inhibitors.
  37. JUMPshiny: A User-Friendly Platform for Comprehensive Analysis and Visualization of Quantitative Proteomics Data.
  38. The nuclear import receptor importin-7 targets HPV from the Golgi to the nucleus to promote infection.
  39. The intrinsic properties of mRNAs define their translation efficiency at ribosome shortage.
  40. Anthracyclines attenuate Nrf1-dependent proteolytic pathways and potentiate proteasome inhibitor cytotoxicity.
  41. A cytoplasmic motif in HLA-E that drives clathrin-mediated endocytosis and VCP-associated postendocytic trafficking.
  42. HEIP1 orchestrates pro-crossover protein activity during mammalian meiosis.
  43. Nanoluc oligoproteins as a model system for protein misfolding and refolding studies.
  44. Global comparative structural analysis of responses to protein phosphorylation.
  45. MDM2 promotes CELF6 ubiquitination-dependent degradation to promote neuroblastoma cell proliferation.
  46. Cryo-EM structural analyses reveal plant-specific adaptations of the CDC48 unfoldase.
  1. Autophagy. 2025 Nov;21(11): 2311-2312
    A paradoxical role of autophagy: promoting protein aggregates and hindering rejuvenation in a Caenorhabditis elegans L1 arrest model.
    Yuxiang Huang, Daniel J Klionsky.
      Macroautophagy (hereafter referred to as autophagy) is widely recognized as a central pathway for the clearance of protein aggregates and the maintenance of proteostasis. However, a recent study by Murley et al. challenges this conventional view. Using a Caenorhabditis elegans L1 arrest aging model, the authors found that autophagy activation impedes rejuvenation by promoting the accumulation of intra- 10 lysosomal protein aggregates and inducing lysosomal membrane damage. This unexpected finding reveals that autophagy may play dual, context-dependent roles in proteostasis, acting not only as a protective mechanism but also, under certain conditions, as a contributor to cellular stress.
    Keywords:  Aging; autophagy; protein aggregates; proteostasis; rejuvenation; stress
    DOI:  https://doi.org/10.1080/15548627.2025.2541430
  2. Nat Struct Mol Biol. 2025 Oct 20.
    Global analysis of translocon remodeling during protein synthesis at the ER.
    Arunkumar Sundaram, Qianru Li, Yu Wan, Josephine Tang, Haoxi Wu, Luka Smalinskaitė, Ramanujan S Hegde, Zhe Ji, Robert J Keenan.
      Protein biogenesis at the endoplasmic reticulum requires translocons comprising the Sec61 protein-conducting channel and several dynamically associated accessory factors. Here we used transcriptome-wide selective ribosome profiling in human cells to monitor cotranslational interactions of accessory factors for N-glycosylation (the OST-A complex) and multipass membrane protein synthesis (the GEL, PAT and BOS complexes). OST-A was preferentially recruited to open Sec61 channels engaged in polypeptide translocation; conversely, GEL, PAT and BOS were recruited synchronously to closed Sec61 channels and stabilized by newly inserted transmembrane domains. Translocon composition changed repeatedly and reversibly during the synthesis of topologically complex multipass membrane proteins. These data establish the molecular logic that underlies substrate-driven translocon remodeling, events that are crucial for the efficient biogenesis of secretory and membrane proteins.
    DOI:  https://doi.org/10.1038/s41594-025-01691-6
  3. Mol Cell. 2025 Oct 23. pii: S1097-2765(25)00817-2. [Epub ahead of print]
    A structural basis for chaperone repression of stress signaling from the endoplasmic reticulum.
    Lisa Neidhardt, Joanne Tung, Miriam Kuchersky, Jakub Milczarek, Vasileios Kargas, Katherine Stott, Rina Rosenzweig, David Ron, Yahui Yan.
      The endoplasmic reticulum (ER) unfolded protein response (UPR) is tuned by the balance between unfolded proteins and chaperones. Reserve chaperones suppress UPR transducers via their stress-sensing luminal domains, but the underlying mechanisms remain unclear. The ER chaperone AGR2 is known to repress the UPR transducer IRE1β. Here, structural prediction, X-ray crystallography, and NMR spectroscopy identify critical interactions between an AGR2 monomer and a regulatory loop in IRE1β's luminal domain. However, in the repressive complex, it is an AGR2 dimer that binds IRE1β. Cryoelectron microscopy (cryo-EM) reconstruction explains this feature: one AGR2 protomer engages the regulatory loop, while the second asymmetrically binds IRE1β's luminal domain's C terminus, blocking IRE1β-activating dimerization. Molecular dynamic simulations indicate that the second, disruptive AGR2 protomer exploits rare fluctuations in the IRE1β dimer that expose its binding site. Thus, AGR2 disrupts IRE1β dimers to suppress the UPR, priming the system for activation by chaperone clients that compete for AGR2.
    Keywords:  endoplasmic reticulum; intestinal mucin; molecular chaperones; protein multimerization; signal transduction; transmembrane protein; unconventional splicing; unfolded protein response
    DOI:  https://doi.org/10.1016/j.molcel.2025.09.032
  4. Biochem Soc Trans. 2025 Oct 22. pii: BST20253074. [Epub ahead of print]
    The many faces of the GID/CTLH E3 ligase complex.
    Arno F Alpi, Jakub Chrustowicz, Dawafuti Sherpa, Brenda A Schulman.
      The GID/C-terminal to LisH (CTLH) E3 is an emerging family of evolutionarily conserved multiprotein E3 ligase complexes implicated in various biological processes including metabolic rewiring, stress-responsive regulation, cellular differentiation, and immunity. Pioneering biochemical reconstitution, cryo-EM, and cell-based studies have illuminated many aspects of the compositional and structural dynamics of GID/CTLH E3 complexes. GID/CTLH E3 undergoes sophisticated regulation through incorporation of interchangeable substrate receptors and association with supramolecular assembly factors enabling higher-order complex formation. Furthermore, paralogous subunits vary and may modulate function across cell types. Additionally, an assortment of regulatory factors fine-tune substrate selection, underscoring the adaptability of this E3 ligase system. Here, we review these distinct ubiquitin ligase features, examine the mechanistic implications of GID/CTLH E3 regulation and the exquisite targeting of oligomeric substrates, and discuss potential for therapeutic application in targeted protein degradation.
    Keywords:  E3 ubiquitin ligase; GID/CTLH E3; degron; higher-order E3 assembly; molecular glue; oligomeric metabolic enzymes; ubiquitin proteasome system
    DOI:  https://doi.org/10.1042/BST20253074
  5. Cell Rep. 2025 Oct 16. pii: S2211-1247(25)01218-5. [Epub ahead of print]44(10): 116447
    Heat shock induces silent ribosomes and reorganizes mRNA turnover.
    Sayanur Rahaman, Nicole Schiffelholz, Nitish Mittal, Klemens E Fröhlich, Mihaela Zavolan, Attila Becskei.
      mRNAs associate with single or multiple ribosomes; these ribosomal assemblies-monosomes and polysomes-translate the mRNAs before degradation. The impact of heat stress on this mRNA turnover remains unclear. We show that in heat-shocked yeast cells, the proportion of monosomes increases without a corresponding rise in the number of associated mRNAs. Consequently, most monosomes are devoid of mRNAs and silent, lacking translational initiation factors and proteins facilitating posttranslational folding. Such silent monosomes also appear under other stress conditions, with proportions varying according to stress type, suggesting that they represent a general feature of cellular adaptation. In parallel with the induction of silent ribosomes, elevated temperatures reduce the overall rate of mRNA-ribosome association with few exceptions. Notably, heat shock promotes the ribosomal association of transcripts encoding heat shock proteins, without extension of the half-lives of these mRNAs. These mechanisms dynamically reorganize mRNA turnover to prioritize the translation of heat shock proteins over other proteins.
    Keywords:  4-thiouracil; CP: Genomics; CP: Molecular biology; Saccharomyces cerevisiae; codon optimality; mRNA half-life; mRNA metabolic labelling; macrophage; polysome profiling; proteomics; stress granule; xrn1
    DOI:  https://doi.org/10.1016/j.celrep.2025.116447
  6. Redox Biol. 2025 Oct 08. pii: S2213-2317(25)00397-0. [Epub ahead of print]87 103884
    The mitochondrial disulphide relay substrate FAM136A safeguards IMS proteostasis and cellular fitness.
    Christine Zarges, Hanna Fieler, Robin Alexander Rothemann, Simon Poepsel, Lucas T Jae, Jan Riemer.
      The mitochondrial disulphide relay is the key machinery for import and oxidative protein folding in the mitochondrial intermembrane space. Among IMS proteins with unknown function, we identified FAM136A as a new substrate of the mitochondrial disulphide relay. We demonstrate a transient interaction between FAM136A and MIA40, and that MIA40 introduces four disulphide bonds in two twin-CX3C motifs of FAM136A. Consequently, IMS import of FAM136A requires these cysteines and its steady state levels in intact cells are strongly dependent on MIA40 and AIFM1 levels. Furthermore, we show that FAM136A forms non-covalent homodimers as a mature protein. Acute deletion of FAM136A curtails cellular proliferation capacity and elicits a robust induction of the integrated stress response, coincident with the aggregation and/or depletion of selected IMS proteins including HAX1 and CLPB. Together, this establishes FAM136A as a pivotal component of the IMS proteostasis network, with implications for overall cellular function and health.
    Keywords:  FAM136A; Integrated stress response; MIA40; Oxidative protein folding
    DOI:  https://doi.org/10.1016/j.redox.2025.103884
  7. Nat Commun. 2025 Oct 20. 16(1): 8966
    Optineurin is an adaptor protein for ubiquitinated substrates in Golgi membrane-associated degradation.
    Yoichi Nibe-Shirakihara, Shinya Honda, Satoko Arakawa, Satoru Torii, Hajime Tajima Sakurai, Hirofumi Yamaguchi, Shigeru Oshima, Ryuichi Okamoto, Michael Lazarou, Hideshi Kawakami, Shigeomi Shimizu.
      Golgi membrane-associated degradation (GOMED) is a process that leading to the degradation of proteins that have passed through the trans-Golgi membranes upon Golgi stress. GOMED is morphologically similar to autophagy, but the substrates degraded are different, and they thus have different biological roles. Although the substrate recognition mechanism of autophagy has been clarified in detail, that of GOMED is completely unknown. Here we report that GOMED degrades its substrate proteins selectively via optineurin (OPTN), as we found that the degradation of GOMED substrates is s`uppressed by the loss of OPTN. OPTN binds to K33 polyubiquitin-tagged proteins that have passed through the Golgi, which are then incorporated into GOMED structures for eventual degradation. In vivo, GOMED is known to be involved in the removal of mitochondria from erythrocytes, and in Optn-deficient mice, mitochondria are not degraded by GOMED, resulting in the appearance of erythrocytes containing mitochondria. These findings provide insight into the substrate recognition mechanism of GOMED.
    DOI:  https://doi.org/10.1038/s41467-025-64400-3
  8. Signal Transduct Target Ther. 2025 Oct 23. 10(1): 358
    Navigating the landscape of protein folding and proteostasis: from molecular chaperones to therapeutic innovations.
    Omer Faruk Kuzu, Lars Jørgen Tvenge Granerud, Fahri Saatcioglu.
      Protein folding is a fundamental process ensuring that polypeptide chains acquire the correct three-dimensional structures required for biological function. This complex journey from nascent polypeptides to mature proteins is tightly regulated by the cellular proteostasis network-an integrated system of molecular chaperones, folding enzymes, and degradation machineries. Disruptions in this network lead to dysproteostasis, a pathological state implicated in a growing list of human diseases, including neurodegenerative disorders, metabolic syndromes, and cancer. In this review, we provide a comprehensive and multidimensional analysis of protein folding biology, tracing its evolution from early theoretical foundations to cutting-edge biophysical and computational techniques that now permit near-atomic-resolution modeling of folding dynamics. We explore the historical progression of protein folding research, including landmark discoveries of secondary structure, chaperone biology, and energy landscape theory. We detail the roles of key molecular chaperones across cytosolic, mitochondrial, and endoplasmic reticulum compartments, emphasizing their collaborative actions in protein folding and quality control. We also discuss the multifactorial causes of protein misfolding-from genetic mutations to aging and oxidative stress-and examine the pathological consequences, paying special attention to diseases characterized by toxic protein aggregation and loss of proteome fidelity. We then examine therapeutic innovations targeting proteostasis, including chaperone modulators, proteostasis pathway inhibitors, and emerging strategies to increase proteome resilience. By consolidating insights at the molecular, cellular, and systems levels, this review underscores the central role of protein folding homeostasis in health and disease and highlights novel opportunities for therapeutic intervention through the modulation of the proteostasis network.
    DOI:  https://doi.org/10.1038/s41392-025-02439-w
  9. Dev Cell. 2025 Oct 20. pii: S1534-5807(25)00570-2. [Epub ahead of print]60(20): 2701-2702
    ATG9 assists lipid replenishment for lysosome membrane repair.
    Hemmo Meyer, Bojana Kravic.
      Lysosomal membranes can be permeabilized under various conditions with detrimental consequences for the cell. In this issue, de Tito et al. report that the lipid scramblase ATG9, best known for its role in autophagosome formation, helps distribute lipids from the ER to reseal the limiting membrane and restore lysosomal function.
    DOI:  https://doi.org/10.1016/j.devcel.2025.09.010
  10. Mol Cell. 2025 Oct 20. pii: S1097-2765(25)00815-9. [Epub ahead of print]
    Delayed protein translocation protects mitochondria against toxic CAT-tailed proteins.
    Nils Bertram, Toshiaki Izawa, Felix Thoma, Serena Schwenkert, Stéphane Duvezin-Caubet, Sae-Hun Park, Nikola Wagener, Anne Devin, Christof Osman, Walter Neupert, Dejana Mokranjac.
      Ribosome-associated protein quality control (RQC) protects cells against the toxic effects of faulty polypeptides produced by stalled ribosomes. However, mitochondria are vulnerable to C-terminal alanyl and threonyl (CAT)-tailed proteins that are generated in this process, and faulty nuclear-encoded mitochondrial proteins are handled by the recently discovered mitoRQC. Here, we performed a genome-wide screen in yeast to identify additional proteins involved in mitoRQC. We found that peptidyl-tRNA hydrolase 2 (Pth2), present in the mitochondrial outer membrane, influences aggregation of CAT-tailed proteins without majorly affecting the CAT-tailing process itself. Peptidyl-tRNA hydrolase activity is essential during this process, yet the activity of Pth2 can be substituted by another peptidyl-tRNA hydrolase upon proper localization. Our data suggest that Pth2 acts by modulating protein translocation and that the mitochondrial proteostasis network is relieved through increased access of CAT-tailed proteins to cytosolic chaperones. Other hits obtained in the screen show that, in general, delayed protein translocation protects mitochondria against toxic CAT-tailed proteins.
    Keywords:  RQC; TOM complex; cellular homeostasis; mitoRQC; mitochondria; peptidyl-RNA hydrolase; protein translocation
    DOI:  https://doi.org/10.1016/j.molcel.2025.09.030
  11. Sci Adv. 2025 Oct 24. 11(43): eadw6064
    In situ characterization of mitochondrial Hsp60-Hsp10 chaperone complex under folding stress.
    Mingyu Jung, Minjung Kim, Su Jin Ham, Jongkyeong Chung, Soung-Hun Roh.
      Mitochondrial proteostasis is critical for maintaining mitochondrial function, and its disruption induces mitochondrial unfolded protein response, which up-regulates chaperones to alleviate protein-folding stress. However, how these chaperones mitigate protein-folding stress remains unclear. Here, using correlated cryo-electron tomography, we show that folding stress triggers marked mitochondrial morphological changes, including the accumulation of amorphous protein aggregates and increased abundance and spatial clustering of the mitochondrial heat shock protein 60-heat shock protein 10 (mtHsp60-Hsp10) complex. Subtomogram analysis revealed the in situ architecture and conformational heterogeneity of mtHsp60-Hsp10 under stress, which retains its canonical double-ring structure while adopting distinct football, half-football, and bullet-like states. Notably, the mtHsp60-Hsp10 complex encapsulates unstructured substrates through conserved hydrophobic interactions. We further demonstrate that knockdown of the mtHsp60-Hsp10 complex exacerbates folding stress, as evidenced by elevated cellular stress responses and activation of mitophagy. Our study defines the in situ structural properties of the mtHsp60-Hsp10 complex and provides mechanistic insight into how it safeguards mitochondrial proteostasis under folding stress.
    DOI:  https://doi.org/10.1126/sciadv.adw6064
  12. Essays Biochem. 2025 Oct 22. pii: EBC20253026. [Epub ahead of print]
    Small-molecule degron mimetics for targeted protein degradation.
    Xingui Liu.
      More than 80% of intracellular proteins are degraded by the ubiquitin-proteasome system. This system relies on a cascade of enzymes-E1 (ubiquitin-activating enzyme), E2 (ubiquitin-conjugating enzyme), and E3 (ubiquitin ligase)-to catalyze the polyubiquitination of target proteins, which are then recognized and degraded by the 26S proteasome. Among these enzymes, E3 ubiquitin ligases play a central role by specifically recognizing degron motifs on substrate proteins. The presence and accessibility of these degrons often dictate the half-life and stability of a given protein. Leveraging this mechanism, the artificial introduction of degrons or degron mimetics into otherwise stable proteins has emerged as a novel strategy in drug discovery for selectively degrading disease-causing proteins. In this short review, I will highlight small-molecule degron mimetics that have been developed for targeted protein degradation.
    Keywords:  C-degron; N-degron; cyclic imide degron; degron; degron mimetics; hydroxyproline degron; phosphodegron
    DOI:  https://doi.org/10.1042/EBC20253026
  13. Blood. 2025 Oct 20. pii: blood.2024026749. [Epub ahead of print]
    The Proteostasis Network is a Therapeutic Target in Acute Myeloid Leukemia.
    Kentson Lam, Yoon Joon Kim, Evelyn Li-Ting Tan, Carlo Miguel Ong, Andrea Zoey Liu, Fanny Jiahua Zhou, Mary Jean Sunshine, Bernadette Chua, Silvia Vicenzi, Katelyn Zhining Chen, Helena Yu, Pierce Ford, Jie-Hua Zhou, Yuning Hong, Eric J Bennett, Leslie A Crews, Edward D Ball, Robert Signer.
      Oncogenic growth places great strain and dependence on protein homeostasis (proteostasis). This has made proteostasis pathways attractive therapeutic targets in cancer, but efforts to drug these pathways have yielded disappointing clinical outcomes. One exception is proteasome inhibitors, which are approved for frontline treatment of multiple myeloma. However, proteasome inhibitors are largely ineffective for treatment of other cancers at tolerable doses, including acute myeloid leukemia (AML), although reasons for these differences are unknown. Here, we determined that proteasome inhibitors are ineffective in AML due to inability to disrupt proteostasis. In response to proteasome inhibition, AML cells activated HSF1 and increased autophagic flux to preserve proteostasis. Genetic inactivation of HSF1 sensitized AML cells to proteasome inhibition, marked by accumulation of unfolded protein, activation of the PERK-mediated integrated stress response, severe reductions in protein synthesis, proliferation and cell survival and significant slowing of disease progression and extension of survival in vivo. Similarly, combined autophagy and proteasome inhibition suppressed proliferation, synergistically killed human AML cells, and significantly reduced AML burden and extended survival in vivo. Furthermore, autophagy and proteasome inhibition preferentially suppressed protein synthesis and colony formation, and induced apoptosis in primary patient AML cells, including AML stem/progenitor cells, compared to normal hematopoietic stem/progenitor cells. Combined autophagy/proteasome inhibition activated a terminal integrated stress response, which was surprisingly driven by Protein kinase R (PKR). These studies unravel how proteostasis pathways are co-opted to promote AML growth, progression and drug resistance, and reveal that disabling the proteostasis network is a promising strategy to therapeutically target AML.
    DOI:  https://doi.org/10.1182/blood.2024026749
  14. Nat Commun. 2025 Oct 22. 16(1): 9333
    Computational design and evaluation of optimal bait sets for scalable proximity proteomics.
    Vesal Kasmaeifar, Saya Sedighi, Anne-Claude Gingras, Kieran R Campbell.
      The spatial organization of proteins within eukaryotic cells underlies essential biological processes and can be mapped by identifying nearby proteins using proximity-dependent biotinylation approaches such as BioID. When applied systematically to hundreds of bait proteins, BioID has localized thousands of endogenous proteins in human cells, generating a comprehensive view of subcellular organization. However, the need for large bait sets limits the scalability of BioID for context-dependent spatial profiling across different cell types, states, or perturbations. To address this, we develop a benchmarking framework with multiple complementary metrics to assess how well a given bait subset recapitulates the structure and coverage of a reference BioID dataset. We also introduce GENBAIT, a genetic algorithm-based method that identifies optimized bait subsets predicted to retain maximal spatial information while reducing the total number of baits. Applied to three large BioID datasets, GENBAIT consistently selected subsets representing less than one-third of the original baits while preserving high coverage and network integrity. This flexible, data-driven approach enables intelligent bait selection for targeted, context-specific studies, thereby expanding the accessibility of large-scale subcellular proteome mapping.
    DOI:  https://doi.org/10.1038/s41467-025-64383-1
  15. J Biol Chem. 2025 Oct 16. pii: S0021-9258(25)02675-4. [Epub ahead of print] 110823
    SDF2 and SDF2L1 are essential co-factors of DNAJB11 for Polycystin-1 processing.
    Tilman Busch, Björn Neubauer, Sophia Sediq, Paul Christoph Zeisler, Stefan Tholen, Oliver Schilling, Lukas Westermann, Michael Köttgen.
      Mutations in the co-chaperone DNAJB11 have been shown to cause polycystic kidney disease. The molecular mechanism underlying DNAJB11-related kidney disease involves impaired processing of Polycystin-1 (PC1), the protein most commonly mutated in autosomal dominant polycystic kidney disease (ADPKD). Chaperones are known to form multi-protein complexes to facilitate folding and processing of client proteins. Yet, it is unknown whether DNAJB11 forms complexes with other proteins that are required for PC1 processing. In this study, we perform an unbiased interaction proteomics screen for DNAJB11-interacting proteins. We identify two highly homologous proteins, SDF2 and SDF2L1, as strong interaction partners of DNAJB11. Using newly established knockout cell lines, we demonstrate a reciprocal interdependence of DNAJB11 and SDF2/SDF2L1 protein abundance. Furthermore, we show that concomitant loss of SDF2 and SDF2L1 impairs PC1 processing, mimicking the biochemical phenotype caused by loss of DNAJB11. Using a combination of knockout cell lines and re-expression of the respective members of the DNAJB11 protein complex, we show that SDF2 or SDF2L1 are elementary subunits of the DNAJB11 complex required for normal PC1 processing.
    DOI:  https://doi.org/10.1016/j.jbc.2025.110823
  16. Sci Adv. 2025 Oct 24. 11(43): eado4330
    ITCH regulates Golgi integrity and proteotoxicity in neurodegeneration.
    Qiwang Xiang, Yuning Lu, Hongjin Wang, Hao Chen, Peng Chen, Xiaofeng Zhao, Larissa Cortes Morales, Yiwei Yang, Junqin Yang, Tao Zhang, Jiou Wang.
      Golgi fragmentation is an early and common feature of neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS) and Alzheimer's disease (AD). However, whether a shared mechanism drives Golgi fragmentation across different neurodegenerative conditions remains unclear. Here, we identify the E3 ubiquitin-protein ligase Itchy homolog (ITCH) as a key regulator of proteotoxicity through its role in inducing Golgi fragmentation. Disease-associated accumulation of ITCH promotes fragmentation of both the cis- and trans-Golgi networks, disrupting protein sorting and impairing lysosomal functions. The ITCH-dependent lysosomal dysfunction compromises the clearance of misfolded proteins associated with several neurodegenerative diseases. Inhibition of ITCH protects against proteotoxicity in both mammalian neurons and Drosophila models of neurodegeneration. The accumulation of ITCH in patients with ALS and AD is attributed to up-regulation of the ubiquitin-specific protease USP11, which deubiquitinates and stabilizes ITCH. These results uncover a pathogenic pathway regulating Golgi integrity and contributing to the development of neurodegenerative diseases.
    DOI:  https://doi.org/10.1126/sciadv.ado4330
  17. Nat Struct Mol Biol. 2025 Oct 24.
    RNase MRP subunit composition and role in 40S ribosome biogenesis.
    Eric M Smith, Jimmy Ly, Sofia Haug, Iain M Cheeseman.
      RNase MRP and RNase P are evolutionarily related complexes that facilitate rRNA and tRNA biogenesis, respectively. The two enzymes share nearly all protein subunits and have evolutionarily related catalytic RNAs. Notably, RNase P includes a unique subunit, RPP21, whereas no RNase MRP-specific proteins have been found in humans, limiting molecular analyses of RNase MRP function. Here, we identify the RNase MRP-specific proteins, C18orf21 (RMP24) and NEPRO (RMP64). C18orf21/RMP24 and RPP21 display significant structural homology, but we identify specific regions that drive interactions with their respective complexes. By targeting these RNase MRP-specific subunits, our functional analysis reveals that RNase MRP is essential for rRNA processing and preferentially required for 40S ribosome biogenesis. Finally, we determine that disease-associated mutations in RMP64 impair its association with RNase MRP subunits. Together, our findings elucidate the molecular determinants of RNase MRP function and underscore its critical role in ribosome biogenesis and disease.
    DOI:  https://doi.org/10.1038/s41594-025-01690-7
  18. EMBO J. 2025 Oct 20.
    Reticulophagy receptor FAM134C restrains BMP receptor signaling.
    Shuchen Gu, Hanchenxi Zhang, Jin Cao, Zhou He, Jianfeng Wu, Xia Liu, Mingjie Zheng, Ting Liu, Bin Zhao, Pinglong Xu, Qiming Sun, Jianping Jin, Xia Lin, Yi Yu, Jiahuai Han, Xin-Hua Feng.
      FAM134/RETREG family members are ER-phagy receptors that maintain cellular homeostasis by regulating endoplasmic reticulum turnover. However, possible non-ER-phagy functions of FAM134 proteins remain elusive. Here, we show that RETREG3/FAM134C functions as a selective autophagy receptor for the type I BMP receptor (BMPRIA/ALK3) and recruits BMPRIA into LC3-containing autophagosomes for subsequent degradation. FAM134C-induced degradation diminishes the availability of BMP receptors and thus the strength of BMP signaling. Inhibition of autophagy through chemical means or knockdown of key autophagy regulators, ATG5 or Beclin-1, prevents BMPR1A degradation. Additionally, disruption of the putative LC3-interacting region (LIR) motif in FAM134C completely abolishes its interaction with LC3, thereby impeding its ability to degrade BMPR1A. Moreover, FAM134C-deficient mice exhibit enhanced BMP responses in the intestines, which affects intestinal crypt regeneration. Our findings suggest that FAM134C acts as a specific receptor that controls BMP signaling through the autophagic degradation of the type I BMP receptor, independent of its canonical role in ER-phagy.
    Keywords:  Autophagy; Degradation; RETREG; Smad; TGF-β
    DOI:  https://doi.org/10.1038/s44318-025-00581-3
  19. J Biol Chem. 2025 Oct 22. pii: S0021-9258(25)02699-7. [Epub ahead of print] 110847
    Mammalian and Bacterial Adaptors Function as Co-Disinhibitory Pairs to Activate the E3 ubiquitin ligase WWP2.
    Samkeliso V Blundell, Mei Liu, Romina Tocci, Andrea Majstorovic, Solène Tsang, David W Holden.
      The NEDD4-like E3 ubiquitin ligase, WWP2, is involved in a range of host processes from cell differentiation to T cell immunity. Ligase activity is tightly regulated with WWP2 being held in an autoinhibited state. Binding of a PY motif-containing adaptor, an Ndfip, via the WW domains of NEDD4-like E3 ubiquitin ligases leads to their disinhibition. Here, we show that the canonical Ndfip, NDFIP2, requires multiple PY motifs for interaction with and activation of WWP2. In contrast, the single PY-motif containing Ndfips TMEM127 and SUSD6 function as a co-disinhibitory pair. TMEM127 and the Salmonella protein SteD also function as a co-disinhibitory pair. However, SteD requires a different region of WWP2, the C2 domain, for interaction with WWP2 and this interaction results in disinhibition of WWP2. These findings demonstrate a range of ways that Ndfips can disinhibit WWP2. To our knowledge, these are the first examples of two Ndfips functioning as co-disinhibitory pairs, and of a bacterial effector that disinhibits an E3 ubiquitin ligase.
    Keywords:  Salmonella enterica; adaptor protein/ ubiquitin; virulence factor/ E3 ubiquitin ligase
    DOI:  https://doi.org/10.1016/j.jbc.2025.110847
  20. Autophagy. 2025 Oct 20.
    The Drosophila ZER1 homolog interacts with ref(2)P to regulate autophagy and Keap1-cnc/NFE2L2/Nrf2-mediated oxidative stress.
    Yi-Ting Wang, Ya-Ting Shen, Hsuan-Yu Weng, Jung-Kun Wen, Guang-Chao Chen.
      The ubiquitin-proteasome system (UPS) and macroautophagy/autophagy are two major pathways for maintaining cellular protein homeostasis. Increasing evidence has highlighted the complex interactions and crosstalk between these pathways; however, the specific molecules and mechanisms mediating the interplay between the UPS and autophagy are still not fully elucidated. In this study, we discovered that knocking down the Drosophila Cul2 (Cullin 2)-RING ubiquitin ligase complex adaptor CG12084/DmZer1 impedes autophagy and autophagic flux. DmZer1 interacts with the Drosophila SQSTM1/p62 homolog ref(2)P, promoting its association with ubiquitinated proteins and degradation. ref(2)P is a crucial player in regulating autophagy and the Keap1-cnc/NFE2L2 pathway-mediated antioxidant response. Knockdown of DmZer1 leads to the formation of ref(2)P bodies, which sequester Keap1 and promote cnc/NFE2L2-mediated antioxidant responses under oxidative stress conditions. These findings reveal the pivotal role of DmZer1 in regulating autophagy and the ref(2)P-Keap1-cnc/NFE2L2-mediated oxidative stress response.
    Keywords:  Drosophila; SQSTM1/p62; ZER1; autophagy; oxidative stress; ubiquitin-proteasome system
    DOI:  https://doi.org/10.1080/15548627.2025.2577771
  21. Biophys J. 2025 Oct 22. pii: S0006-3495(25)00694-0. [Epub ahead of print]
    Plasma membrane-endoplasmic reticulum coupling probed with genetically-encoded voltage sensors.
    Masoud Sepehri Rad, Meyer B Jackson.
      The endoplasmic reticulum (ER) forms an elaborate contiguous network extending through the cytoplasm of eukaryotic cells. The ER is surrounded by a membrane that separates its lumen from the cytoplasm. The ER membrane harbors channels and pumps capable of controlling ion flux and creating a voltage gradient. Because the ER membrane potential is difficult to study experimentally little is known about how voltage influences its many vital functions. Here we introduce optical probes of ER membrane potential derived from the hybrid voltage sensor (hVoS) family of genetically-encoded voltage sensors. Probes were targeted to the ER using motifs from three ER proteins, Sec61β, cytochrome P450, and cytochrome b5 type A. As shown recently with other types of ER voltage sensors, patch-clamp fluorometry recording with our new probes demonstrated that voltage steps applied to the plasma membrane elicit a voltage change at the ER membrane. These probes exhibited subtle differences in their responses suggesting they target different ER compartments. The steeper voltage dependence of Sec61β-hVoS (mCerulean3-Sec61β) signals suggested that this probe targets an ER compartment rich in voltage-gated ion channels. The ER voltage change is slow, but its onset is virtually synchronous with the plasma membrane voltage step. This suggests a direct electrical coupling into the ER lumen through plasma membrane-ER contacts. Analysis with the aid of an equivalent circuit provided an estimate of the resistance of these contacts. The rapid, direct transmission of voltage changes from the plasma membrane to the ER provides a mechanism for regulating ER function that could be especially important in excitable cells. The sensors introduced here provide researchers with powerful tools for imaging ER voltage and assessing its impact on cellular function.
    DOI:  https://doi.org/10.1016/j.bpj.2025.10.023
  22. EMBO J. 2025 Oct 22.
    Functional landscape of ubiquitin linkages couples K29-linked ubiquitylation to epigenome integrity.
    Javier Arroyo-Gomez, Matthew J Murray, Claire Guérillon, Juanjuan Wang, Ekaterina Isaakova, Nazaret Reverón-Gómez, Mikaela Koutrouli, Aldwin Suryo Rahmanto, Katrine Mitrofanov, Andreas Ingham, Sofie Schovsbo, Katrine Weischenfeldt, Fabian Coscia, Dimitris Typas, Moritz Völker-Albert, Victor Solis, Lars Juhl Jensen, Anja Groth, Andreas Mund, Petra Beli, Robert F Shearer, Niels Mailand.
      Linkage-specific ubiquitin chains govern the outcome of numerous critical ubiquitin-dependent signaling processes, but their targets and functional impacts remain incompletely understood due to a paucity of tools for their specific detection and manipulation. Here, we applied a cell-based ubiquitin replacement strategy enabling targeted conditional abrogation of each of the seven lysine-based ubiquitin linkages in human cells to profile system-wide impacts of disabling formation of individual chain types. This revealed proteins and processes regulated by each of these poly-ubiquitin topologies and indispensable roles of K48-, K63- and K27-linkages in cell proliferation. We show that K29-linked ubiquitylation is strongly associated with chromosome biology, and that the H3K9me3 methyltransferase SUV39H1 is a prominent cellular target of this modification. K29-linked ubiquitylation catalyzed by TRIP12 and reversed by TRABID constitutes the essential degradation signal for SUV39H1 and is primed and extended by Cullin-RING ubiquitin ligase activity. Preventing K29-linkage-dependent SUV39H1 turnover deregulates H3K9me3 homeostasis but not other histone modifications. Collectively, these data resources illuminate cellular functions of linkage-specific ubiquitin chains and establish a key role of K29-linked ubiquitylation in epigenome integrity.
    Keywords:  Epigenome maintenance; Heterochromatin; Linkage-specific ubiquitylation; Ubiquitin; Ubiquitin-dependent proteolysis
    DOI:  https://doi.org/10.1038/s44318-025-00599-7
  23. Oncogene. 2025 Oct 21.
    On-genetic inactivation of caspase-3 and P53 increases cancer cell fitness by PDIA4 redistribution.
    Gal Twito, Faiza Amterat Abu Abayed, Ayelet Gilad, Suma Biadsy, Noa Gavriel, Suad Sheikh Suliman, Yarden Mizrahi, Hila Megged, Mor Tenenboim, Naim Abu-Freha, Aeid Igbaria.
      Numerous cellular pathways are known to cause resistance in cancer cells. The unfolded protein response (UPR), a signaling pathway activated during proteostasis stress in the endoplasmic reticulum (ER), is an adaptive process to increase cancer cell fitness. However, the molecular mechanism between ER stress, UPR activation, and chemoresistance is insufficiently understood. Here, we report that ER stress induction and UPR activation are necessary for chemoresistance to cisplatin and doxorubicin. Mild ER stress is a sufficient precondition for cancer cells to evade cisplatin- and doxorubicin-associated cell death. Mechanistically, ER stress induction results in the redistribution of PDIA4 from the ER to the cytosol, facilitated by the c-tail-anchored proteins DNAJB12 and DNAJB14 and the cytosolic HSC70-cochaperone SGTA. In the cytosol, PDIA4 forms an inhibitory interaction with caspase-3 and wt-p53, leading to their attenuation and increased cancer cell proliferation. Furthermore, we show that PDIA4 must originate from the ER to inhibit caspase-3 and wt-p53 in the cytosol. Silencing PDIA4, DNAJB12/14, or SGTA rescues wt-p53 and caspase-3 activity. Finally, we found that in tumors isolated from colorectal cancer patients, PDIA4 and DNAJB12 are highly expressed compared to their healthy tissues; this expression is associated with the induction of the UPR. Our data show a novel non-genetic mechanism to inhibit apoptosis and suggest PDIA4, DNAJB12/14, and SGTA as novel therapeutic targets to rescue apoptosis and inhibit proliferation in cancer cells.
    DOI:  https://doi.org/10.1038/s41388-025-03606-7
  24. Nat Commun. 2025 Oct 20. 16(1): 9257
    Enhanced hybridization-proximity labeling discovers protein interactomes of single RNA molecules.
    Karen Yap, Tek Hong Chung, Erin C Hedges, Agnes L Nishimura, Christopher E Shaw, Eugene V Makeyev.
      RNAs engage diverse protein partners and localize to specific subcellular compartments, yet dissecting proteomes associated with low-abundance or dispersed RNA molecules remains a challenge. We present an enhanced hybridization-proximity labeling (HyPro) technology for in situ proteome profiling of endogenously expressed RNA microcompartments. We re-engineer the HyPro enzyme and optimize proximity biotinylation conditions to identify proteins associated with compact RNA-containing nuclear bodies, small pre-mRNA clusters, and individual transcripts. Applying this approach to pathogenic G4C2 repeat-containing C9orf72 RNAs, retained as single-molecule foci in the nuclei of amyotrophic lateral sclerosis (ALS) patient-derived pluripotent stem cells, we reveal extensive interactions with disease-linked paraspeckle markers and a specific set of pre-mRNA splicing factors. These findings highlight early RNA processing and localization defects in ALS that may contribute to this late-onset neurodegenerative disorder. Overall, HyPro provides a broadly applicable platform for mapping RNA-protein interactions, enabling insights into RNA biology and its dysregulation in disease.
    DOI:  https://doi.org/10.1038/s41467-025-64282-5
  25. EMBO J. 2025 Oct 20.
    TXNIP mediates LAT1/SLC7A5 endocytosis to limit amino acid uptake in cells entering quiescence.
    Jennifer Kahlhofer, Nikolas Marchet, Kristian Zubak, Brigitta Seifert, Madlen Hotze, Anna-Sophia Egger-Hörschinger, Lucija Kucej, Claudia Manzl, Yannick Weyer, Sabine Weys, Martin Offterdinger, Sebastian Herzog, Veronika Reiterer, Chiara Volani, Marcel Kwiatkowski, Saskia B Wortmann, Siamak Nemati, Johannes A Mayr, Johannes Zschocke, Bernhard Radlinger, Kathrin Thedieck, Leopold Kremser, Bettina Sarg, Lukas A Huber, Hesso Farhan, Mariana E G de Araujo, Susanne Kaser, Sabine Scholl-Bürgi, Daniela Karall, David Teis.
      Entry into and exit from cellular quiescence require dynamic adjustments in nutrient acquisition, yet the mechanisms by which quiescent cells downregulate amino acid (AA) transport remain poorly understood. Here we show that cells entering quiescence selectively target plasma membrane-resident amino acid transporters for endocytosis and lysosomal degradation. This process matches amino acid uptake with reduced translational demand and promotes survival during extended periods of quiescence. Mechanistically, we identify the α-arrestin TXNIP as a key regulator of this metabolic adaptation, since it mediates the endocytosis of the SLC7A5-SLC3A2 (LAT1-4F2hc) AA transporter complex in response to reduced AKT signaling. To promote transporter ubiquitination, TXNIP interacts with NEDD4L and other HECT-type ubiquitin ligases. Loss of TXNIP disrupts this regulation, resulting in dysregulated amino acid uptake, sustained mTORC1 signaling, and ultimately cell death under prolonged quiescence. The characterization of a novel TXNIP loss-of-function variant in a patient with a severe metabolic disease further supports its role in nutrient homeostasis and human health. Together, these findings highlight TXNIP's central role in controlling nutrient acquisition and metabolic plasticity with implications for quiescence biology and diseases.
    Keywords:  Amino Acids Uptake; Endocytosis; Quiescence; SLC7A5/LAT1; TXNIP
    DOI:  https://doi.org/10.1038/s44318-025-00608-9
  26. RSC Med Chem. 2025 Oct 21.
    Targeted protein degradation of HSP90 and associated proteins for cancer therapy via PROTACs and beyond.
    Gulshan Kumar, Gouri Tyagi, Vikramdeep Monga.
      In recent years, heat shock protein 90 (HSP90), a widely expressed molecular chaperone, has emerged as a promising anticancer target due to its crucial role in stabilizing and regulating the functions of numerous client proteins involved in various essential cellular processes, including protein folding, signalling pathways, and activation of tumor-associated proteins. Despite extensive developments, only one HSP90 inhibitor has gained approval, reflecting the complexity of the HSP90 chaperone machinery, associated side effects, and emergence of resistance mechanisms. To overcome these limitations, researchers have focused their attention on developing targeted protein degraders (TPDs), a revolutionary therapeutic approach that selectively eliminates specific dysregulated target proteins. TPDs exploit cellular degradation pathways, including the ubiquitin-proteasome system (UPS), lysosomal pathways, and autophagy to achieve precise protein degradation. Among these strategies, proteolysis-targeting chimeras (PROTACs) as well as HEMTAC/HIM-PROTACs have emerged as prominent UPS-based technologies. PROTACs link targets to E3 ligases for proteasomal removal, where HEMTACs exploit HSP90 to drive client ubiquitination, thereby offering significant potential for cancer therapeutics. Given HSP90's role in tumor progression and considering the potential of TPDs, researchers have designed and developed various HSP90-targeting PROTACs and HEMTAC/HIM-PROTACs, which exhibits remarkable efficacy, selectivity, antiproliferative potency, and the ability to overcome drug resistance. This review highlights the structural and biological functions of HSP90, delineates the mechanistic principles underlying its degradation, and summarizes the structure-activity relationships (SARs) inlcuding the synthetic strategies employed across different HSP90-directed TPD modalities. Furthermore, the challenges and opportunities associated with the utilization of HSP90 and their client proteins in developing TPDs-based therapeutics to tackle the unmet clinical needs in cancer have been discussed.
    DOI:  https://doi.org/10.1039/d5md00369e
  27. Nat Commun. 2025 Oct 20. 16(1): 9275
    Physics-based evolution of transmembrane helices reveals mechanisms of cholesterol attraction.
    Jeroen Methorst, Nino Verwei, Christian Hoffmann, Paweł Chodnicki, Roberto Sansevrino, Partha Pyne, Han Wang, Niek van Hilten, Dennis Aschmann, Alexander Kros, Loren Andreas, Jacek Czub, Dragomir Milovanovic, Herre Jelger Risselada.
      The existence of linear cholesterol-recognition motifs in transmembrane domains has long been debated. Evolutionary molecular dynamics (Evo-MD) simulations-genetic algorithms guided by (coarse-grained) molecular force-fields-reveal that thermodynamic optimal cholesterol attraction in isolated alpha-helical transmembrane domains occurs when multiple consecutive lysine/arginine residues flank a short hydrophobic segment. These findings are supported by atomistic simulations and solid-state NMR experiments. Our analyses illustrate that linear motifs in transmembrane domains exhibit weak binding affinity for cholesterol, characterized by sub-microsecond residence times, challenging the predictive value of linear CRAC/CARC motifs for cholesterol binding. Membrane protein database analyses suggest even weaker affinity for native linear motifs, whereas live cell assays demonstrate that optimizing cholesterol binding restricts transmembrane domains to the endoplasmic reticulum post-translationally. In summary, these findings contribute to our understanding of cholesterol-protein interactions and offer insight into the mechanisms of protein-mediated cholesterol regulation within membranes.
    DOI:  https://doi.org/10.1038/s41467-025-63769-5
  28. Proc Natl Acad Sci U S A. 2025 Oct 28. 122(43): e2516849122
    Nir2 crystal structures reveal a phosphatidic acid-sensing mechanism at ER-PM contact sites.
    Dongyoung Kim, Seowhang Lee, Youngsoo Jun, Changwook Lee.
      Agonist-induced activation of phosphoinositide-specific phospholipase C (PLC) converts phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] to diacylglycerol (DAG) at the inner leaflet of the plasma membrane (PM). DAG can be enzymatically transformed into phosphatidic acid (PA) and accumulated at the PM. PYK2 N-terminal domain-interacting receptor 2 (Nir2) mediates the formation of ER-PM membrane contact sites (MCSs) by specifically recognizing PA at the PM and directly interacting with ER membrane protein vesicle-associated membrane protein-associated proteins (VAPs). The N-terminal phosphatidylinositol transfer protein domain of Nir2 facilitates PI/PA exchange at ER-PM MCSs to maintain PI and PA levels. Here, we reveal the mechanisms by which Nir2 senses phosphatidic acid (PA) and associates with membranes, based on three crystal structures of its C-terminal Lipin/Ned1/Smp2 (LNS2) domain bound to PA, the diphenylalanine [FF]-containing acidic tract (FFAT) motif complexed with vesicle-associated membrane protein-associated protein B/C (VAPB), and the Asp-Asp-His-Asp (DDHD) domain. The C-terminal LNS2 domain of Nir2 directly interacts with the phosphate in the headgroup of PA via hydrogen bonds involving S1025, T1065, K1103, and K1126. Formation of a salt bridge between E355 in Nir2 and R55 in VAPB is essential for Nir2 FFAT-VAPB interaction. The central DDHD domain of Nir2 forms a twofold symmetric dimer, and this self-association contributes to stable and tight membrane association. These findings reveal how Nir2-mediated ER-PM MCS formation maintains continued PI(4,5)P2-dependent PLC signaling.
    Keywords:  Nir2; PLC signaling; crystal structure; lipid transfer protein; membrane contact site
    DOI:  https://doi.org/10.1073/pnas.2516849122
  29. EMBO Mol Med. 2025 Oct 21.
    ER stress induced mitochondrial dysfunction drives Treg instability in coronary artery disease.
    Smriti Parashar, Mohammad Oliaeimotlagh, Payel Roy, Qingkang Lyu, Anusha Bellapu, Mikhail Fomin, Sunil Kumar, Yan Wang, Chantel C McSkimming, Coleen A McNamara, Klaus Ley.
      Under conditions of chronic unresolved inflammation characteristic of atherosclerosis, regulatory CD4+ T cells (Tregs) become unstable and convert to cytotoxic exTregs. The mechanism driving this conversion in humans is unclear. Here, we show unresolved endoplasmic reticulum (ER) stress as a key factor driving Treg instability. Human exTregs undergo ER stress and consequent mitochondrial dysfunction that remains unchecked due to defective mitophagy. Integrated stress response (ISR), a pathway that can trigger inflammatory signaling, is also upregulated in exTregs. exTregs are highly apoptotic and are more susceptible to stress-mediated cellular dysfunction due to their senescent state. In a phenotype reminiscent of exTregs, Tregs from coronary artery disease (CAD) patients show high ER stress and mitochondrial depolarization. This is further exacerbated in CD4+ T cells residing in atherosclerotic plaques. Pro-atherosclerotic stressors such as oxLDL and interferon-γ induce ER stress and mitochondrial dysfunction in Tregs in vitro. We conclude that the maladaptive inflammatory environment in atherosclerosis triggers ER stress and mitochondrial dysfunction, contributing to Treg instability in CAD.
    Keywords:  Atherosclerosis; ER Stress; Mitochondrial Dysfunction; Tregs; exTregs
    DOI:  https://doi.org/10.1038/s44321-025-00322-3
  30. iScience. 2025 Oct 17. 28(10): 113612
    Unveiling Sec31: The regulator of COPII vesicle-dependent secretion and beyond.
    Jiaming Nie, Jingzhi Ma.
      Sec31, the outer protein of the coat protein complex II (COPII) vesicle, is recognized as a key regulator of the dynamics of COPII assembly and disassembly, thereby regulating the transport of newly synthesized cargo from the endoplasmic reticulum (ER) to the Golgi complex. In the context of large cargo, such as collagen, Sec31 is capable of modulating the flexibility of the outer shell to ensure precise transportation. In addition to its role in secretion, Sec31 is implicated in various physiological and pathological processes, including autophagy and responses to microbial infections. This review summarizes the functions of Sec31 and its molecular mechanisms.
    Keywords:  cell biology; molecular biology
    DOI:  https://doi.org/10.1016/j.isci.2025.113612
  31. J Biol Chem. 2025 Oct 22. pii: S0021-9258(25)02706-1. [Epub ahead of print] 110854
    J-domain proteins cooperate with Hsp70 to drive multiphase separation of RNA-binding-deficient TDP-43.
    Kian Hua Yeo, Jian Hua Kong, Qing Hao Ng, Mi-Jeong Yoon, Olivia Agatha, Eunyoung Chae, Hyun O Lee, H Shawn Je, Young-Jun Choe.
      RNA-free TDP-43, resulting from mutations or post-translational modifications in its RNA-binding domain, forms multiphase condensates with Hsp70 chaperones enriched in the core. The presence of this structure in the nucleus is thought to be associated with disease states. However, the mechanisms underlying its formation remain poorly understood. In particular, it is unclear whether J-domain proteins (JDPs), critical co-chaperones of Hsp70, are incorporated into the multiphase condensates, and if so, how they contribute to the phase separation process. Using yeast as a model organism with a relatively small JDP family, we identified Sis1, but not Ydj1, as an important factor in TDP-43 multiphase separation. RNA-binding-deficient TDP-43 initially forms uniform condensates enriched with the JDP Sis1 but not with Hsp70. Subsequent recruitment of Hsp70 transforms these uniform structures into multiphase condensates, with both Sis1 and Hsp70 enriched in the core. This transition requires a functional J-domain, which stimulates Hsp70 ATPase activity. These findings reveal a key role for JDPs in TDP-43 multiphase separation and highlight JDP specificity in this process.
    Keywords:  J-domain protein (JDP); RNA binding protein; Saccharomyces cerevisiae; TAR DNA‐binding protein 43 (TDP‐43); amyotrophic lateral sclerosis (ALS); heat shock protein 40 (Hsp40); heat shock protein 70 (Hsp70); molecular chaperone; phase separation
    DOI:  https://doi.org/10.1016/j.jbc.2025.110854
  32. EMBO Rep. 2025 Oct 20.
    Tudor-based proteomic strategy pan-specifically enriches and identifies protein arginine methylation.
    Lingzi Lu, Ting Li, Rou Zhang, Yutong Wang, Xiaoping Ye, Yixin Luo, Lingyu Sun, Liang Qi, Zilu Ye, Yang Mao, Yanqiu Yuan.
      Protein arginine methylation is an important post-translational modification (PTM) in eukaryotes, regulating a variety of biological processes. Proteomic profiling of arginine methylation has advanced our understanding of its roles in biology and disease. However, pan-specific enrichment of methylarginine-containing peptides remains challenging. Herein we report a molecular affinity strategy based on the Tudor domain of SMN, a naturally occurring methylarginine reader protein, for comprehensive proteomic profiling of cellular arginine methylation. We demonstrate that the Tudor domain-based approach exhibits broad specificity for proteins harboring mono- or di-methylated arginines, encompassing both RGG/RG-rich and non-RG motifs, facilitating the discovery of novel methylation sites. Using this strategy, we identify asymmetric dimethylarginine (aDMA) in protein eIF3D, an essential component of the eukaryotic translation initiation complex. Biochemical analyses reveal that aDMA modification at R99 of eIF3D plays a regulatory role in protein translation initiation. Our findings establish a generally applicable approach for proteomic profiling of arginine methylation and unveil its novel regulatory role for this modification in eukaryotic protein translation.
    Keywords:  Arginine Methylation; Molecular Affinity Enrichment; Proteomics; eIF3D
    DOI:  https://doi.org/10.1038/s44319-025-00599-y
  33. Autophagy. 2025 Oct 23.
    Human clearance systems have a layered architecture across tissues and cell types that supports varied proteome compositions.
    Ekaterina Vinogradov-Talyah, Bar Edri, Lior Ravkaie, Or Lazarescu, Fadi Gharra, Juman Jubran, Anat Ben-Zvi, Esti Yeger-Lotem.
      Protein clearance is fundamental to proteome health. In eukaryotes, it is carried out by two highly conserved proteolytic systems, the ubiquitin-proteasome system (UPS) and the autophagy-lysosome pathway (ALP). Despite their pivotal role, the basal organization of the human protein clearance systems across tissues and cell types remains uncharacterized. Here, we interrogated this organization using diverse omics datasets. Relative to other protein-coding genes, UPS and ALP genes were more widely expressed, encoded more housekeeping proteins, and were more essential for growth, in accordance with their fundamental roles. Nevertheless, UPS and ALP subsystems had varied expression patterns. Furthermore, each system showed a layered organization. The larger layer included genes that were differentially expressed across tissues; tissue-specific upregulation was associated with tissue-specific functions, phenotypes, and disease susceptibility. The second smaller layer included genes that were stably expressed across tissues, more highly and widely expressed, had more protein interactions, and were more essential for growth, suggesting that they act as a core. Last, we compared protein clearance to other branches of the proteostasis network. Protein clearance and folding were closely coordinated across tissues and more plastic than protein synthesis. Taken together, we propose that the proteostasis network is organized hierarchically and is tailored to the proteome composition. This organization could contribute to and illuminate tissue-selective phenotypes.
    Keywords:  Autophagy-lysosome pathway (ALP); Mendelian diseases; protein clearance; proteostasis; tissue-specific; ubiquitin-proteasome system (UPS)
    DOI:  https://doi.org/10.1080/15548627.2025.2580035
  34. BMC Biol. 2025 Oct 21. 23(1): 315
    Establishment of an imaging-based screening pipeline for the identification of human ribosome biogenesis inhibitors.
    Claudia Gafko, Réka Hollandi, Kerstin Dörner, Matteo Rosellini, Ivo Zemp, Peter Horvath, Ulrike Kutay.
       BACKGROUND: Ribosomes are huge ribonucleoprotein particles that mediate protein synthesis in all organisms. The synthesis of ribosomes is a complex process that involves hundreds of supporting factors in mammalian cells, including proto-oncogenes and tumor suppressors. Dysregulation of ribosome biogenesis can contribute to tumorigenesis, and the increased production of ribosomes in cancer cells is known to promote proliferative cell growth. Therefore, ribosome biogenesis represents an attractive vulnerability of cancer cells that ought to be exploited for the development of anti-cancer drugs. Despite the large number of trans-acting factors promoting ribosome assembly including potentially druggable enzymes, only few chemical inhibitors that act on ribosome biogenesis, especially downstream of pre-rRNA transcription, have been identified to date.
    RESULTS: To enable large-scale screens for chemical compounds that interfere with ribosome biogenesis, we have established a pipeline to perform single-cell, imaging-based screening campaigns using four different readouts, including fluorescent ribosomal protein reporters (RPS2-YFP, RPL29-GFP) and immunofluorescence analyses of the ribosome biogenesis factor ENP1(BYSL), in HeLa cells, a human cancer line. We have assessed the robustness of our high-content screening approach by performing a pilot screen using a library comprising more than 1000 FDA-approved drugs with known targets in other pathways. This pilot screen obtained excellent quality scores and identified ten compounds as hits. These hit compounds likely affect ribosome synthesis indirectly, the majority by inducing DNA damage or by inhibiting the proteasome. We therefore used the identified compounds to establish appropriate counter assays for DNA damage and proteasome inhibition, to exclude common indirect effects in the downstream analysis of such screening campaigns.
    CONCLUSIONS: The established screening pipelines provide a robust, efficient, and sensitive experimental framework to identify chemical compounds that impair ribosome synthesis. The combination of readouts allows to distinguish effects on pre-rRNA synthesis from downstream effects on ribosome assembly. Established counter assays on DNA damage and protein degradation enable to exclude effects on these pathways, which commonly interfere with ribosome synthesis indirectly. The developed assays are easily scalable to screen libraries of higher complexity in the future.
    Keywords:  Cancer; Chemical compound screen; Drug; High-content screening; Image analysis; Machine learning; Microscopy; Ribosomal subunit; Ribosome synthesis; rRNA
    DOI:  https://doi.org/10.1186/s12915-025-02425-2
  35. Nucleic Acids Res. 2025 Oct 22. pii: gkaf1039. [Epub ahead of print]
    CIRCpedia v3: an interactive database for circular RNA characterization and functional exploration.
    Si-Nan Zhai, Yu-Yao Zhang, Mo-Han Chen, Zhi-Can Fu, Ling-Ling Chen, Xu-Kai Ma, Li Yang.
      Recent advances on genome-wide profiling and characterization of circular RNAs have suggested their versatile roles in diverse biological processes, yet systematic elucidation of their molecular characteristics and functional mechanisms remains challenging. Here, we introduce CIRCpedia v3 (https://bits.fudan.edu.cn/circpediav3), an expanded repository to annotate both circular RNAs from back-splicing of exons (circRNAs) and circular RNAs from intron lariats (ciRNAs) by profiling 2413 sequencing datasets across 20 species. Building upon the previous version of CIRCpedia, this release identifies >2 million circular RNAs and introduces transformative advances to facilitate circular RNA research: (i) community-recommended nomenclature with enhanced molecular profiling, enabling quantitative comparison of circular/linear isoform dynamics; (ii) an interactive platform with real-time comparative analyses of circRNAs and visualizations; and (iii) integrated toolkits to identify base-editable sites, predict circRNA subcellular localization, detect circRNA degradation signals for stability optimization, predict m6A modification sites, assess circRNA coding potential, and design divergent polymerase chain reaction primers and small interfering RNAs (siRNAs). By integrating insights from cross-species expression, molecular characterization, and functional predictions, CIRCpedia v3 empowers researchers to prioritize context-specific circular RNA candidates in biological or disease conditions and to accelerate mechanistic discovery and therapeutic development.
    DOI:  https://doi.org/10.1093/nar/gkaf1039
  36. Proc Natl Acad Sci U S A. 2025 Oct 28. 122(43): e2515947122
    Adipose cullin 3 mediates the antiobesity effect of pan neddylation inhibitors.
    Lijie Gu, Yanhong Du, Mohammad Nazmul Hasan, Yung-Dai Clayton, Tiangang Li.
      Cullin Ring E3 Ligases (CRLs) belong to the largest family of multisubunit ubiquitin E3 ligases. A cullin serves as the scaffold protein that recruits E3 ligases and substrate receptors in a CRL complex, whose activity requires cullin neddylation, a posttranslational modification that can be pharmacologically targeted by neddylation inhibitors. Elevated neddylation activity has been observed in the liver and adipose tissue of obese mice, implicating a pathogenic link between altered CRL activity and the development of metabolic disorders. Emerging evidence has also shown that neddylation inhibitors possess antiobesity and hypoglycemic property. However, the roles of cullin proteins in regulating adipocyte biology are still incompletely defined. Here, we report that pan neddylation inhibitor TAS4464 treatment reversed obesity and adipose inflammation, resulting in improved hepatic steatosis and insulin sensitivity in obese mice. Among all mammalian cullin proteins that were targeted by TAS4464, we identified that cullin 3 (Cul3) was required for adipogenesis and adipocyte hypertrophy. A complete absence of Cul3 in adipocytes caused severely inhibited adipose expansion associated with ectopic fat accumulation in the liver and brown adipose tissue and insulin resistance, while adipocyte-specific Cul3 haploinsufficiency attenuated obesity and improved overall metabolic homeostasis, which recapitulated the metabolic benefits of TAS4464. Mechanistically, we found that Cul3 inhibition caused adipose nuclear factor erythroid 2-related factor 2 (NRF2) stabilization, which contributed to impaired adipogenesis by inhibiting lipogenesis. Together, these findings demonstrate that Cul3 is required during adipogenesis and acts as a downstream mediator of the antiobesity effect of pan neddylation inhibitors.
    Keywords:  adipogenesis; diabetes; insulin resistance; obesity
    DOI:  https://doi.org/10.1073/pnas.2515947122
  37. Proteomics. 2025 Oct 21. e70061
    JUMPshiny: A User-Friendly Platform for Comprehensive Analysis and Visualization of Quantitative Proteomics Data.
    Aijun Zhang, Yingxue Fu, Zuo-Fei Yuan, Long Wu, Dehui Kong, Ling Li, Zhiping Wu, Pjotr Prins, Junmin Peng, Xusheng Wang.
      Mass spectrometry-based quantitative proteomics has revolutionized our understanding of biological processes and unveiled the molecular mechanisms underlying various diseases. The analysis and visualization of quantitative proteomics data remain complex and require user-friendly tools with robust analytical capacities. In this study, we introduce JUMPshiny, a novel, interactive, and comprehensive web-service, that is built on R-Shiny and designed for processing and presenting quantitative proteomics data. JUMPshiny includes a wide range of visualizations and offers a streamlined workflow, including experimental design, data exploration, batch normalization, differential analysis, and enrichment analysis. Through examples, we demonstrate automated quality control, interactive data visualization, and customizable statistical analyses. Built on the R-Shiny framework, JUMPshiny integrates established libraries and packages to ensure computational robustness and reproducibility. Overall, JUMPshiny represents a powerful platform for proteomics data analysis for the research community. JUMPshiny is available at https://jumpshiny.genenetwork.org. The source code is available under MIT license at: https://github.com/Wanglab-UTHSC/JUMP_shiny.
    Keywords:  JUMPshiny; R packages; bioinformatics tools; data analysis; data visualization; mass spectrometry; quantitative proteomics; software; web application
    DOI:  https://doi.org/10.1002/pmic.70061
  38. Sci Adv. 2025 Oct 24. 11(43): eadz6792
    The nuclear import receptor importin-7 targets HPV from the Golgi to the nucleus to promote infection.
    Tai-Ting Woo, Yuka Takeo, Mara C Harwood, Ethan T Houck, Daniel DiMaio, Billy Tsai.
      During human papillomavirus (HPV) entry, the virus exploits COPI-dependent retrograde transport to cross the Golgi apparatus before reaching the nucleus to cause infection. How HPV enters the nucleus after exiting the Golgi is unclear, although mitotic nuclear envelope breakdown (NEB) appears important. Here, we show that importin-7 (IPO7), a nuclear pore import receptor, is present at the Golgi and promotes HPV infection. IPO7 knockdown inhibits infection and causes HPV to accumulate in the Golgi without reaching mitotic chromosomes, demonstrating that IPO7 promotes Golgi-to-nucleus transport of HPV. Golgi-to-nucleus transport of a cellular cargo also requires IPO7, suggesting that HPV hijacks a preexisting pathway for nuclear entry. Furthermore, the C-terminal nuclear localization sequence of HPV L2 protein, which overlaps its cell-penetrating peptide sequence, binds IPO7 directly in a COPI-dependent virus trafficking step. Together, these data identify a role for an importin in HPV infection and suggest that the canonical nuclear pore import machinery plays an unanticipated role in NEB-dependent nuclear entry.
    DOI:  https://doi.org/10.1126/sciadv.adz6792
  39. Nucleic Acids Res. 2025 Oct 14. pii: gkaf1013. [Epub ahead of print]53(19):
    The intrinsic properties of mRNAs define their translation efficiency at ribosome shortage.
    Elena S Babaylova, Alexander V Gopanenko, Alexey E Tupikin, Marsel R Kabilov, Alexey A Malygin.
      A deficiency of ribosomal proteins is a severe stress to the cell. Haploinsufficiency of human ribosomal protein genes, including RPS26, is the cause of ribosomopathies. Here, we reduced the level of ribosomal protein eS26 in HEK293T cells, which caused a decrease in the level of 40S ribosomal subunits and led to a ribosome-shortage state. We show that eS26-deficient cells have lighter polysomes than control cells. Using RNA-sequencing of total and polysome-associated messenger RNA (mRNA) fractions from both cell types, we identify thousands of differentially expressed genes in the transcriptome and translatome, respectively, as well as genes with altered translation efficiency. By analyzing the intrinsic properties of the mRNAs of these genes, we demonstrate that under ribosome-deficient conditions, the translation efficiency of mRNAs that have longer coding sequences, lower GC levels, and higher abundance increases, whereas that of mRNAs with opposite characteristics decreases. We provide a mathematical rationale that describes changes in the translatome at a ribosome-deficient state through alterations in the rate of translation initiation and density of ribosomes on mRNA. We propose that, in ribosome deficiency, the decrease in the translation efficiency of mRNAs of genes critical for cell differentiation, such as GATA1, is determined by the intrinsic properties of these mRNAs.
    DOI:  https://doi.org/10.1093/nar/gkaf1013
  40. Mol Biol Cell. 2025 Oct 22. mbcE25040183
    Anthracyclines attenuate Nrf1-dependent proteolytic pathways and potentiate proteasome inhibitor cytotoxicity.
    Janakiram R Vangala, Madison A Ward, Bader Albalawi, Ahmed M Elshazly, Nayyerehalsadat Hosseini, Shanwei Shen, Holly A Byers, Christiane Carter, Jinze Liu, Senthil K Radhakrishnan.
      Proteasome inhibitors such as bortezomib, carfilzomib, and ixazomib are FDA-approved treatments for multiple myeloma, but resistance frequently limits their effectiveness. The transcription factor Nrf1 (NFE2L1) upregulates proteasome and autophagy genes upon proteasome inhibition, contributing to adaptive resistance. In this study, we identified anthracyclines, including doxorubicin, as suppressors of the Nrf1-driven transcriptional response. Mechanistically, doxorubicin impaired Nrf1 binding to antioxidant response elements (AREs) within promoter regions of target genes without affecting Nrf1 processing or nuclear localization. Importantly, aclarubicin, a non-DNA-damaging anthracycline, also attenuated Nrf1 transcriptional activity, indicating that DNA damage is not required for this inhibition. Doxorubicin co-treatment delayed proteasome recovery after pulse inhibition and partially restored sensitivity to carfilzomib in bortezomib-resistant U266 myeloma cells, consistent with genetic knockout of Nrf1. These findings identify a DNA-damage-independent mechanism by which anthracyclines directly obstruct Nrf1-mediated transcriptional induction. Thus, anthracyclines serve as chemical tools to probe the molecular control of proteostasis and suggest a strategy to mitigate Nrf1-driven adaptive response to proteasome inhibition.
    DOI:  https://doi.org/10.1091/mbc.E25-04-0183
  41. Proc Natl Acad Sci U S A. 2025 Oct 28. 122(43): e2514956122
    A cytoplasmic motif in HLA-E that drives clathrin-mediated endocytosis and VCP-associated postendocytic trafficking.
    Wanlin He, Andreas Damianou, Iolanda Vendrell, Klaus Früh, Daowen I Yin, Frances M Brodsky, Benedikt M Kessler, Simon Brackenridge, Persephone Borrow, Geraldine M Gillespie, Andrew J McMichael.
      Human Leukocyte Antigen E (HLA-E) is a nonclassical MHC class I molecule that exhibits dual immunological functions in regulating natural killer (NK) cells and T cells through unusual trafficking patterns. We previously reported that HLA-E surface expression is low and transient due to its cytoplasmic tail and dominant VL9 peptide, making it a dynamic indicator of cellular status for NK cell surveillance. Here, we identify a sequence motif in the HLA-E cytoplasmic tail that enables rapid internalization via clathrin-mediated endocytosis (CME) through interaction with the adaptor protein 2 (AP-2) complex. Following internalization, HLA-E is routed to endosomes, where the same cytoplasmic motif and peptide loading together facilitate its reappearance on the cell surface-a process influenced by valosin-containing protein (VCP). Our findings reveal previously unrecognized endosomal trafficking pathways and regulatory mechanisms that distinguish HLA-E from classical HLA class I molecules, with broad implications for understanding the immunoregulatory roles of HLA-E.
    Keywords:  HLA-E; clathrin-mediated endocytosis; cytoplasmic tail; nonclassical MHC class I; valosin-containing protein (VCP)
    DOI:  https://doi.org/10.1073/pnas.2514956122
  42. Proc Natl Acad Sci U S A. 2025 Oct 28. 122(43): e2515747122
    HEIP1 orchestrates pro-crossover protein activity during mammalian meiosis.
    Arnaud De Muyt, Sunkyung Lee, Sushil Khanal, Laurine Dal Toe, Céline Adam, Raphael Mercier, Valérie Borde, Neil Hunter, Thomas Robert.
      Meiotic crossovers (COs) are needed to produce genetically balanced gametes. In mammals, CO formation is mediated by a conserved set of pro-CO proteins via mechanisms that remain unclear. Here, we characterize a mammalian pro-CO factor HEIP1. In mouse HEIP1 is essential for crossover and fertility of both sexes. HEIP1 promotes crossover by orchestrating the recruitment of other pro-CO proteins, including the MutSγ complex (MSH4-MSH5) and E3 ligases (HEI10, RNF212, and RNF212B), that are required to mature CO sites and recruit the CO-specific resolution complex MutLγ. Moreover, HEIP1 directly interacts with HEI10, suggesting a direct role in controlling the recruitment of pro-CO E3 ligases. During early stages of meiotic prophase I, HEIP1 interacts with the chromosome axes, independently of recombination, before relocalizing to the central region of the synaptonemal complex. We propose that HEIP1 is a conserved master regulator of CO proteins that controls different CO maturation steps.
    Keywords:  E3-ligases; HEIP1; crossovers; homologous recombination; meiosis
    DOI:  https://doi.org/10.1073/pnas.2515747122
  43. Biophys J. 2025 Oct 22. pii: S0006-3495(25)00696-4. [Epub ahead of print]
    Nanoluc oligoproteins as a model system for protein misfolding and refolding studies.
    Grigory V Mechetin, Piotr E Marszalek.
      Protein misfolding can lead to protein malfunction, which may compromise cell viability. Chaperones, including the HSP70 system, are proteins that have evolved to restore the native structure of misfolded proteins. Although most chaperones, including DnaK (bacterial HSP70), were first described over 30 years ago, important questions related to their mechanisms remain unanswered. Only a small number of model proteins are used in the literature for misfolding and refolding studies. Previously, we described several NanoLuc (Nluc) luciferase-based constructs as models for DnaK-assisted chaperone refolding: Nluc2, Nluc3, and others, where the Nluc module was combined with the titin I91 domain. Here, we expanded this family of tandem multi-modular proteins with Nluc7, which allowed us to better analyze how interactions between modules affect Nlucn activity, denaturation, and DnaK-assisted refolding. We found that interactions between internal modules of Nlucn attenuate the module's activity differently than interactions between terminal and neighboring modules. Also, among the Nlucn variants, Nluc7 is the most resistant to precipitation during thermal denaturation, enabling the production of soluble misfolded proteins at elevated concentrations. After denaturation, Nluc7 show the ability to DnaK-assisted refolding with elevated half-time compared to Nluc2 and Nluc3. The relatively large size of Nluc7 allowed us to characterize both its native and denatured states using transmission electron microscopy, which showed no aggregation but indicated particle compactization after 10 min of denaturation and the formation of small soluble aggregates after 30 min of denaturation.
    Keywords:  DnaK; HSP70; Nanoluc; molecular chaperones; protein misfolding; protein refolding; protein thermal denaturation
    DOI:  https://doi.org/10.1016/j.bpj.2025.10.025
  44. Nat Commun. 2025 Oct 24. 16(1): 9407
    Global comparative structural analysis of responses to protein phosphorylation.
    Miguel Correa Marrero, Victor Hugo Mello, Pablo Sartori, Pedro Beltrao.
      Post-translational modifications (PTMs), particularly protein phosphorylation, are key regulators of cellular processes, impacting numerous aspects of protein activity. Despite widespread phosphorylation of eukaryotic proteomes, the function of most phosphosites remains unknown. Elucidating the structural mechanisms underlying phosphorylation is crucial for understanding its regulatory roles. Here, we present a comparative structural analysis of phosphorylated and non-phosphorylated proteins taken from the Protein Data Bank (PDB). Our study systematically evaluates how phosphorylation affects backbone conformation, protein dynamics, and mechanical strain. We found that phosphorylation commonly induces small, stabilizing conformational changes through conformational selection and frequently modulates local residue fluctuations, influencing overall protein motion. Notably, a small but significant subset of phosphosites shows mechanical coupling with functional sites, aligning with the domino model of allosteric signal transduction. This work provides a foundation for studying phosphorylation and other PTMs in their structural context, which will guide the rational design of synthetic phosphosites and enable the engineering of PTM-driven regulatory circuits in synthetic biology.
    DOI:  https://doi.org/10.1038/s41467-025-64116-4
  45. Cell Death Dis. 2025 Oct 21. 16(1): 736
    MDM2 promotes CELF6 ubiquitination-dependent degradation to promote neuroblastoma cell proliferation.
    Zhenzhen Zhao, Bao Zhang, Xu Zhang, Changchun Li.
      The ubiquitin-proteasome system plays a crucial role in neuroblastoma progression, yet the regulation of key degradation targets remains incompletely understood. By integrating transcriptomic and proteomic data, we identified nine candidate proteins, including CELF6, whose degradation is potentially mediated by ubiquitination. Survival analyses revealed that high CELF6 expression correlated with a favorable prognosis. Functional assays demonstrated that CELF6 suppresses neuroblastoma cell proliferation without affecting apoptosis. Mechanistically, the E3 ubiquitin ligase MDM2 directly interacts with CELF6, promoting its degradation via K48-linked ubiquitination. MDM2 overexpression accelerates CELF6 degradation, while its inhibition stabilizes CELF6 protein levels, an effect reversed by proteasome inhibitors. Furthermore, MDM2-driven neuroblastoma cell proliferation is dependent on CELF6 depletion. These findings establish MDM2 as a key regulator of CELF6 stability and highlight the MDM2-CELF6 axis as a potential therapeutic target in neuroblastoma.
    DOI:  https://doi.org/10.1038/s41419-025-08048-3
  46. Plant Commun. 2025 Oct 23. pii: S2590-3462(25)00334-7. [Epub ahead of print] 101572
    Cryo-EM structural analyses reveal plant-specific adaptations of the CDC48 unfoldase.
    Brandon Huntington, Anandsukeerthi Sandholu, Jun Wang, Junrui Zhang, Lingyun Zhao, Bilal M Qureshi, Umar F Shahul Hameed, Stefan T Arold.
      Targeted protein degradation through the CDC48 unfoldase enables the maintenance and rapid adaptation of proteomes across eukaryotes. However, the profound differences between animals, fungi, and plants are expected to have led to a significant adaptation of the CDC48-mediated degradation. While animal and fungal CDC48 systems have shown structural and functional preservation, such analysis is lacking for plants. We determined the structural and functional characteristics of Arabidopsis thaliana CDC48A in various states and bound to the target-identifying cofactors UFD1 and NPL4. Our analysis reveals several features that distinguish AtCDC48 from its animal and yeast counterparts, despite an 80% sequence identity. Key features are that AtCDC48A displays distinct domain dynamics and interacts differently with AtNPL4. Moreover, AtNPL4 and AtUFD1 do not form an obligate heterodimer, but AtNPL4 can independently bind to AtCDC48A and mediate target degradation; however, their joint action is synergistic. An evolutionary analysis supports that these Arabidopsis features are conserved across plants and represent the ancestral state of eukaryotic CDC48 systems. Jointly, our findings support that plant CDC48 retains a greater modular and combinatorial cofactor usage, highlighting a specific adaptation of targeted protein degradation in plants.
    Keywords:  AAA+ ATPase; Targeted protein degradation; adaptation; cryo-EM; plant protein quality control; ubiquitin-proteasome system
    DOI:  https://doi.org/10.1016/j.xplc.2025.101572
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